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S J Silverstein

Publications and source records attributed to S J Silverstein.

11 recordsLinked to original sources
PubMed1991

The interaction of ICP4 with cell/infected-cell factors and its state of phosphorylation modulate differential recognition of leader sequences in herpes simplex virus DNA.

Regulation of herpes simplex virus (HSV) gene expression requires the synthesis of functional ICP4, a phosphoprotein that binds to several specific sites in virus DNA and acts in trans either to activate or to repress transcription of the three major kinetic classes of virus genes. Binding of ICP4 to specific sites in alpha genes (which are the first to be transcribed) causes repression of alpha-gene expression. ICP4 also indirectly participates in the formation of DNA-protein complexes with sequences present in the promoter/regulatory and leader regions of the beta and gamma genes that are sequentially activated later in infection. Here we demonstrate that the extent of phosphorylation of ICP4 contributes to its ability to participate differentially in complex formation with cis-acting elements present in beta and gamma genes. Dephosphorylated ICP4 retains its binding properties for the high affinity sites present in alpha promoters, whereas only phosphorylated forms of the protein are able to participate in complex formation with model beta and gamma sequences. These studies also reveal a requirement for cell and infected-cell factors to recognize the beta and gamma sequences. Our data suggest that the state of phosphorylation and concentration of ICP4 within the nucleus of infected cells determine the extent to which ICP4 interacts with these other factors.

Acid Phosphatase↗
PubMed1990

Regulation of herpes simplex virus immediate-early gene promoters in mouse neuroblastoma cells.

The non-permissivity of C1300 mouse neuroblastoma cells for herpes simplex virus (HSV) infection is due to a failure of such cells to transcribe the immediate-early (IE) genes following viral infection. We have transfected both C1300 cells and permissive cells with constructs in which each of the 5 IE promoters drives expression of the readily assayable chloramphenicol acetyl transferase (CAT) gene. These experiments show that the lack of IE gene transcription in C1300 cells is due to the weak activity of the five IE promoters in these cells compared to that observed in a range of permissive cell types. This effect is mediated both by up-stream elements and by sequences present in the minimal promoter. The different effects of DNA concentration on the activities of the minimal and complete promoters suggests that the up-stream sequences act by binding a repressor factor present in C1300 cells whilst the weak activity of the minimal promoter results from the absence of a positive factor in such cells.

Animals↗
PubMed1990

Renaturation of denatured lambda repressor requires heat shock proteins.

The temperature-sensitive bacteriophage lambda cI857 repressor protein rapidly renatures after thermal inactivation. E. coli mutants in the heat shock protein genes dnaK, dnaJ, and grpE do not efficiently reactivate heat-denatured repressor. Our results suggest that protein refolding is promoted by heat shock proteins and that such a process is the basis of the homeostatic role played by these proteins in the heat shock response.

Bacteriophage lambda↗
PubMed1990

Interaction of cell and virus proteins with DNA sequences encompassing the promoter/regulatory and leader regions of the herpes simplex virus thymidine kinase gene.

During the course of a productive infection with herpes simplex virus (HSV), gene expression is coordinately regulated in a cascade fashion. Three major kinetic classes of genes, termed alpha, beta, and gamma, are sequentially activated. The mechanism responsible for repression and subsequent activation of beta and gamma genes is not known. A mobility-shift electrophoresis assay was used to examine DNA fragments containing the promoter/regulatory and the mRNA leader regions of the thymidine kinase gene (TK, a model beta gene) for their ability to bind proteins present in nuclear extracts prepared from uninfected and infected cells. Specific complexes unique to each extract were formed. Using a monoclonal antibody specific for ICP4 (the major regulatory protein of HSV) we demonstrated that this protein is present in the complexes formed between probes encompassing either the promoter/regulatory or leader sequence DNAs and proteins in infected-cell extracts. These complexes formed despite the lack of a high affinity binding site for ICP4 in either of these regions. The stability of complexes formed in infected-cell extracts with DNA probes containing the promoter/regulatory, leader region, and a high affinity ICP4-binding site were compared by dissociation analysis. The relative kd(obs) for these DNA-protein complexes was in the order: TK-leader region much greater than TK-promoter/regulatory region greater than or equal to high affinity ICP4-binding site. Cu+/1,10-phenanthroline footprinting revealed that infected-cell complexes which form on a probe containing a high affinity ICP4-binding site generate a protection pattern, whereas those formed on a probe containing the TK-leader sequence do not. In contrast, complexes formed with the latter probe in extracts from uninfected cells are kinetically stable and refractile to cleavage. A model for activation of the TK gene which incorporates these results is presented.

Binding Sites↗
PubMed1990

Characterization of DNA-protein complex formation in nuclear extracts with a sequence from the herpes simplex virus thymidine kinase gene.

The biochemical characteristics of complex formation in nuclear extracts from mock-infected and herpes simplex virus (HSV)-infected Vero and HeLa cells with a sequence downstream of and adjacent to the promoter for the HSV thymidine kinase gene were studied using the mobility shift electrophoresis assay. This region is bound by host cell proteins, as evidenced by the formation of complexes after incubation in extracts from mock-infected cells. Unique virus-specific complexes form in extracts prepared from infected cells, and these complexes contain ICP4, the major regulatory protein of HSV. Examination of the salt requirements for assembly and the stability of preformed DNA-protein complexes to added salt demonstrate the distinct nature of the complexes that form in each extract. This finding is supported by analyses of the relative association and dissociation rates of these complexes which show that complexes formed in extracts prepared from infected cells are kinetically labile. After depletion with chelators, the divalent cation requirements for complex formation were assayed by supplementation with various metal salts. Addition of Mn2+ restored binding activity in extracts from both mock-infected and infected HeLa cells. Finally, footprinting assays revealed that sequences on each strand throughout this region of the thymidine kinase gene were involved in complex formation only in extracts from mock-infected cells. These experiments suggest that one consequence of virus gene expression is to alter the interaction of cell proteins with virus DNA.

Animals↗
PubMed1990

Occurrence of human papillomavirus type 16 DNA in cutaneous squamous and basal cell neoplasms.

Sixty-eight cutaneous squamous cell neoplasms (in situ and invasive) and 26 basal cell carcinomas from 89 patients were analyzed for DNA sequences homologous to the human papillomavirus (HPV) types found predominantly in the genital tract. Thirty-six (53%) of the squamous cell neoplasms contained HPV DNA as detected by filter or in situ hybridization analysis. The frequency of detection of HPV DNA was dependent on the site of the lesion. Of 40 genital squamous cell neoplasms (penile, vulvar, and perianal), 27 (68%) had detectable HPV DNA. In 25 of these, the HPV type was 16 or HPV-16-related, which was similar to the results for the squamous cell neoplasms of the finger (HPV DNA in 9 of 11 tumors with HPV-16 in seven). None of 16 squamous cell neoplasms from sites other than the genital tract or the finger had detectable HPV DNA. HPV DNA was detected in one of the 26 basal cell carcinomas (4%). We conclude that, for cutaneous epithelial malignancies, HPV-16 is restricted to squamous cell neoplasms of the genital tract and finger. These data are consistent with venereal transmission of HPV-16 to the periungual region and suggests a role for this virus in the evolution of squamous cell carcinoma at this site.

Adult↗
PubMed1990

Murine cytotoxic T lymphocytes specific for herpes simplex virus type 1 recognize the immediate early protein ICP4 but not ICP0.

Vaccinia virus recombinants expressing the herpes simplex virus type 1 (HSV-1) genes encoding ICP0 or ICP4 were used to identify the precise target antigen(s) of murine anti-viral cytotoxic T lymphocytes (CTL) specific for the non-structural immediate early proteins. These studies revealed that HSV-1-specific CTL, restricted to class I major histocompatibility complex genes of the H-2k haplotype but not the H-2d or H-2b haplotypes, would lyse autologous cells expressing ICP4. HSV-1-specific CTL derived from various mice strains failed to lyse target cells expressing ICP0. Calculation of the frequencies of H-2k-restricted virus-specific CTL demonstrated that approximately a third of the total HSV-1-specific response was directed against ICP4. Immunization of mice with either recombinant vaccinia virus or transfected L cells expressing ICP4 induced HSV-1-specific lymphoproliferation and delayed hypersensitivity but CTLs were not induced. More importantly, such immunized animals were unable to resist or control a subsequent challenge with virulent HSV-1.

Animals↗
PubMedDate not supplied

Computer-facilitated nutritional analysis.

A seven-day dietary analysis of 59 students in the dental health profession indicated that their nutrient consumption was generally adequate. A dental health examination indicated excellent oral hygiene. Nevertheless, many students were deficient in specific nutrient categories. Results suggested that computerized nutritional analysis techniques have general applicability in dental practice and epidemiological testing.

Ascorbic Acid↗